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目的:探讨PKR通过SUMO化修饰上调P53功能,阐明胰岛β细胞增殖抑制的分子机制。方法:转染wt-PKR质粒并结合BEPP刺激,诱导PKR在胰岛β细胞特异性激活。免疫印迹和免疫共沉淀技术检测P53及P53-SUMO-1蛋白结合水平变化;并给予SUMO化抑制剂Spectomycin B1,分析其相关分子机制。结果:免疫印迹和实时定量PCR检测表明:PKR特异激活能诱导P53蛋白水平而不是mRNA水平上调;免疫共沉淀分析显示:PKR促进了SUMO-1与P53蛋白结合水平的增加;而Spectomycin B1能抑制PKR诱导的P53蛋白水平及其与SUMO结合的增加。结论:PKR能通过促进P53的SUMO化修饰,上调其功能,诱导胰岛β细胞增殖抑制,可能参与2型糖尿病的发生和病程发展。
OBJECTIVE: To explore the molecular mechanism of PKR upregulation of P53 function through SUMOylation and to clarify the inhibition of pancreatic β cell proliferation. Methods: Transfection of wt-PKR plasmid in combination with BEPP stimulation induced specific PKR activation in pancreatic β-cells. Immunoblotting and co-immunoprecipitation were used to detect the changes of P53 and P53-SUMO-1 protein binding. The SUMO inhibitor Spectomycin B1 was also used to analyze the molecular mechanism. Results: Immunoblotting and real-time quantitative PCR showed that PKR-specific activation induced P53 protein level but not mRNA level up-regulation. Co-immunoprecipitation analysis showed that PKR promoted the increase of SUMO-1 and P53 protein binding, while Spectomycin B1 inhibited PKR-induced P53 protein levels and their increased binding to SUMO. Conclusion: PKR can up-regulate the function of P53 by inducing SUMOylation of P53 and induce the inhibition of pancreatic β-cell proliferation, which may be involved in the pathogenesis of type 2 diabetes.